Skip to main content

Main menu

  • For Authors
    • Submit a Manuscript
    • Instructions for Authors
  • Home
  • Content
    • Current Issue
    • Archive
    • Preview Papers
    • Focus Collections
    • Classics Collection
    • Upcoming Focus Issues
  • Advertisers
  • About
    • About the Journal
    • Editorial Board and Staff
  • Subscribers
  • Librarians
  • More
    • Alerts
    • Contact Us
  • Other Publications
    • Plant Physiology
    • The Plant Cell
    • Plant Direct
    • The Arabidopsis Book
    • Plant Cell Teaching Tools
    • ASPB
    • Plantae

User menu

  • My alerts
  • Log in

Search

  • Advanced search
Plant Physiology
  • Other Publications
    • Plant Physiology
    • The Plant Cell
    • Plant Direct
    • The Arabidopsis Book
    • Plant Cell Teaching Tools
    • ASPB
    • Plantae
  • My alerts
  • Log in
Plant Physiology

Advanced Search

  • For Authors
    • Submit a Manuscript
    • Instructions for Authors
  • Home
  • Content
    • Current Issue
    • Archive
    • Preview Papers
    • Focus Collections
    • Classics Collection
    • Upcoming Focus Issues
  • Advertisers
  • About
    • About the Journal
    • Editorial Board and Staff
  • Subscribers
  • Librarians
  • More
    • Alerts
    • Contact Us
  • Follow plantphysiol on Twitter
  • Visit plantphysiol on Facebook
  • Visit Plantae
Research ArticleBREAKTHROUGH TECHNOLOGIES
Open Access

MAPINS, a Highly Efficient Detection Method That Identifies Insertional Mutations and Complex DNA Rearrangements

Huawen Lin, Paul F. Cliften, Susan K. Dutcher
Huawen Lin
Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Paul F. Cliften
Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Paul F. Cliften
Susan K. Dutcher
Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Susan K. Dutcher
  • For correspondence: dutcher@wustl.edu

Published December 2018. DOI: https://doi.org/10.1104/pp.18.00474

  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

  • Tables
  • Additional Files
  • Figure 1.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 1.

    MAPINS identifies flanking genomic DNA sequences around insertion sites. Paired-end 101-bp reads are composed of four different groups. They are reads completely aligned to the Chlamydomonas genome (A, black thin lines); reads completely aligned to the cassette sequence (B, orange thin lines); and reads that are chimeric between Chlamydomonas genomic sequences and the cassette sequence (C and D, mixed black-orange lines). All reads in C have the Chlamydomonas sequence at the 5′ end, and all reads in D have the cassette sequence at the 5′ end. Reads #1 to #6 represent different patterns of chimeric DNA from the 5′ to 3′ end. Step 1, All reads are first aligned to the Chlamydomonas reference genome (black thick line), and all completely aligned reads (group A) are discarded (red dashed box). Two parallel analyses are performed for the remaining reads (groups B, C, and D; red solid box) in steps 2 and 3. Step 2, They are aligned to the cassette sequence (orange thick lines), and all completely aligned reads (group B) are discarded (magenta dashed box). Reads in groups C and D are retained (magenta solid shaded box). Step 3, They are truncated from the 3′ end to 45 bp long in length. These 45-bp reads are aligned to the cassette DNA sequence. Reads that aligned completely (group B and some reads [#4 and #5] in group D) are retained (green solid shaded box). Reads that are not aligned completely are discarded (green dashed box). Step 4, Reads retained from steps 2 (magenta solid box) and 3 (green solid box) are compared. Common retained reads (#4 and #5 in group D) are extracted, and the full-length reads are realigned to the Chlamydomonas reference genome. Breakpoints in these reads define the cassette insertion site (orange vertical line) in the genome.

  • Figure 2.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 2.

    Cosegregation of PCR-validated insertion sites, paroR, and the mutant phenotype. A, Gene structure of the Cre06.g266450 gene. Green box, 5′ Untranslated region (UTR); orange boxes, exons; black lines, introns; purple box, 3′ UTR; blue triangle, insertion site of the cassette; arrows, positions of the primers used in B. The lengths of the blue triangle and arrows are not drawn to scale. B, Insertion of a truncated cassette allows PCR amplification in both mutant and wild-type cells. In six progeny from an octad between 7F3 and CC-125, the primers flanking the Cre06.g266450 insertion sites amplify two different PCR products. In this octad, resistance (R) to paromomycin cosegregates with the larger band (Cre06.g266450+ins [insert]) and sensitivity (S) to paromomycin cosegregates with the smaller band (Cre06.g266450). C, Gene structure of the BAR1 gene. Arrows indicate the positions of the primers used in D. The lengths of the blue triangle and arrows are not drawn to scale. D, The multiciliary phenotype in 1D11 always cosegregates with the insertion in BAR1, as shown in eight random progeny. The absence of a PCR product cosegregates with multiciliary cells (M) and the presence of a PCR product is always found in biciliary cells (B). E, The 1D11 mutant assembles multiple cilia. An antibody to α-tubulin (green) stains cilia protruding outside the round cell body. Bar = 10 µm.

  • Figure 3.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 3.

    Complex DNA rearrangements in multiple insertional strains. A, Diagram of chimeric DNA formed across different chromosomes in five strains (yellow, 9H4; red, 3F8; purple, 8C12; green, 6A12; blue, 9A9). The ends of the arcs indicate the junction sites. Chromosomes 1 to 17 and scaffolds 18 to 54 are drawn to scale in a clockwise direction. B, Positions of the primers in the wild type and in the mutant that form chimeric DNA due to DNA duplication or DNA translocation. Expectations for events on two different chromosomes (A, green; B, magenta) are shown. We assume that the insertion happens on chromosome A and the inserted DNA originates from chromosome B. C, Expected outcome of PCR products from the wild type and mutants with different combinations of primers. D, PCR of wild-type fragments (1F-1R on chromosome 2, 2F-2R on chromosome 8) and a chimeric DNA fragment (1F-2R) in the wild type (CC-124 and CC-125), 8C12, and 9D5. Primers for the mating-type loci (MT, including the MTA1 and MTD1 genes) are included as loading controls. No temp indicates that no DNA template was added to the PCR. E, PCR of wild-type fragments (1F-1R on chromosome 1, 2F-2R on chromosome 12) and a chimeric DNA fragment (1F-2R) in the wild type (CC-124 and CC-125), 8D6, and 9H4.

  • Figure 4.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 4.

    Complex DNA rearrangement adjacent to the APHVIII cassette. A, Diagram of events in 3F8 involving chromosome 2 (green) and chromosome 12 (magenta) in the wild type, chimeric DNA between chromosomes 2 and 12 (#1), between chromosome 2 and the APHVIII cassette (blue; #2), and between chromosome 12 and the APHVIII cassette (#3). The positions of the primers used in B along the chromosomes and the cassette are indicated by short arrows. The orientation of the DNA fragments along the wild-type chromosome (from small to large coordinates) is indicated by long arrows. The diagram is not drawn to scale. A possible DNA rearrangement event in 3F8 is indicated at the bottom. The dashed line indicates the undetermined composition of DNA. B, PCR amplification of multiple DNA fragments in the wild type (CC-124 and CC-125), 3F8, and 3D1. No temp indicates that no DNA template was added to the PCR. C, Diagram of events in 6A12 involving chromosome 5 (green) and chromosome 6 (magenta) in the wild type, chimeric DNA between chromosomes 5 and 6 (#1), between chromosome 5 and the APHVIII cassette (#2), and between chromosome 6 and the APHVIII cassette (#3). The positions of the primers used in D along the chromosomes and the cassette are indicated by short arrows. The orientation of the DNA fragments along the wild-type chromosome (from small to large coordinates) is indicated by long arrows. The diagram is not drawn to scale. A possible DNA rearrangement event in 6A12 is indicated at the bottom. The dashed line indicates the undetermined composition of DNA. D, PCR amplification of multiple DNA fragments in the wild type (CC-124 and CC-125), 6C2, and 6A12. No temp indicates that no DNA template was added to the PCR. E, Diagram of events in 9A9 involving chromosome 9 (green) and chromosome 13 (magenta) in the wild type, chimeric DNA between chromosomes 9 and 13 (#1), between chromosome 9 and the APHVIII cassette (#2), between chromosome 13 and the APHVIII cassette (#3), and between regions of chromosome 13 (#4 and #5). The positions of the primers used in F along the chromosomes and the cassette are indicated by short arrows. The orientation of the DNA fragments along the wild-type chromosome (from small to large coordinates) is indicated by long arrows. The diagram is not drawn to scale. A possible DNA rearrangement event in 9A9 is indicated at the bottom. The dashed line indicates the undetermined composition of DNA. F, PCR amplification of multiple DNA fragments in the wild type (CC-124 and CC-125), 7F3, and 9A9. No temp indicates that no DNA template was added to the PCR.

  • Figure 5.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 5.

    Distribution of 49 new molecular markers for meiotic mapping. Newly designed molecular markers and their positions along different chromosomes are indicated by magenta vertical lines. Their positions along the chromosomes in version 5.5 are indicated above the line. One marker, which maps to both chromosome 12: 5.68 Mb and chromosome 16: 1.21 Mb, is indicated as light blue vertical lines. A subset of previously defined genes used in meiotic mapping is indicated by black vertical lines. Centromeres on each chromosome are indicated as green circles as mapped in Supplemental Table S5. We did not identify centromeres on chromosome 11 or 15.

Tables

  • Figures
  • Additional Files
    • View popup
    Table 1. Breakpoint identification and PCR verification
    Pairs of StrainsChromosomeBreakpoint RangeNo. of ReadsInsertion GapGene IdentifierGene NameMutant in Strain 1Mutant in Strain 2
    bp
    1A4-1D42Unknown–6,876,0171Open endedCre02.g119651Cre02.g119651YesNo
    34,539,389–4,539,406618Cre03.g176651MYSM1NoYes
    174,322,754–4,322,816663Cre17.g730950FLA10YesNo
    2F1-4C216,737,064–6,737,071118Cre01.g048400DZIP1LYesNo
    37,296,662–7,296,6981137Cre03.g206950Cre03.g206950YesNo
    51,027,021–1,027,042422Cre05.g246553Cre05.g246553YesNo
    173,105,419–3,105,4281110Cre17.g721250FAP22NoYes
    3F8-3D114,336,001–4,336,1359135Cre01.g029400TRPC3NoYes
    27,226,958–7,227,7989841Cre02.g145950Cre02.g145950YesNo
    27,290,053–unknown2Open endedCre02.g145500PTK24YesNo
    41,568,203–1,568,5288326Cre04.g213400SUB7YesNo
    104,519,930–4,520,0534124No predicted geneNo predicted geneYesNo
    127,660,573–unknown8Open endedNo predicted geneNo predicted geneYesNo
    17Unknown–768,5663Open endedNo predicted geneNo predicted geneNoNo
    17768,925–unknown7Open endedNo predicted geneNo predicted geneNoNo
    5E3-5F1165,109,304–5,109,339636Cre06.g281050VPS5ANoYes
    92,129,462–2,129,485324Cre09.g393551Cre09.g393551YesNo
    166,582,926–6,582,93085Cre16.g672200BLD11YesNo
    8D6-9H4141,860,469–1,860,4781410Cre14.g620500Cre14.g620500NoYes
    166,583,673–6,583,7081236Cre16.g672200BLD11YesNo
    165,635,438–5,635,438150Cre16.g679550FAP277NoYes
    8C12-9D521,736,814–unknown7Open endedCre02.g086300Cre02.g086300NoYes
    65,398,524–5,398,526133Cre06.g283900TMEM45BYesNo
    96,286,926–6,286,995970Cre09.g406500Cre09.g406500NoYes
    16Unknown–6,571,4441Open endedCre16.g672250MPA13NoNo
    6C2-6A1251,676,418–unknown2Open endedCre05.g242850Cre05.g242850NoYes
    6Unknown–3,512,4121Open endedCre06.g278107Cre06.g278107NoYes
    64,339,867–4,339,9171351Cre06.g278262Cre06.g278262YesNo
    12762,534–unknown2Open endedCre12.g495500Cre12.g495500NoYes
    6F2-6B10161,141,900–unknown2Open endedCre16.g650200MITC17YesNo
    161,266,125–1,266,130156Cre16.g651350ATG11NoYes
    7F3-9A962,254,537–2,254,6121976Cre06.g266450Cre06.g266450YesNo
    61,938,823–1,938,8491027Cre06.g263650Cre06.g263650YesNo
    9Unknown–497,6603Open endedCre09.g404201PHC10NoYes
    133,741,836–unknown3Open endedCre13.g589450Cre13.g589450NoYes
    173,468,906–3,468,91196No predicted geneNo predicted geneNoYes
    1D11-6D11111,722,733–1,722,733210Cre11.g467784SOULYesNo
    161,549,854–1,549,855112Cre16.g653450BAR1YesNo
    17Unknown–5,178,8173Open endedCre17.g735550Cre17.g735550NoYes
    175,244,723–unknown1Open endedCre17.g736000Cre17.g736000NoYes
    17Unknown–5,430,3654Open endedCre17.g736800Cre17.g736800YesNo
    • View popup
    Table 2. Experimentally verified breakpoints by meiotic mapping

    n/a, Not available; ND, not determined.

    No. of InsertsStrainPhenotypeChromosomeGene NameLocation in GeneCosegregation with paroRaCosegregation with PhenotypeaLinkage with paroR
    11D4n/a3MYSM1Intron 95/5n/aYes
    14C2n/a17FAP223′ UTR8/8n/aYes
    15F11n/a6VPS5AExon 311/11n/aYes
    16C2n/a6Cre06.g278262Exon 49/9n/aYes
    16F2Slow growth16MITC17Intron 19/99/9Yes
    16B10n/a16ATG113′ UTR12/12n/aYes
    18C12n/a6TMEM45B5′ UTR16/16n/aYes
    18D6No cilia16BLD11Intron 14 + exon 1524/2424/24Yes
    21A4No cilia17FLA10Exon 1811/1111/11Yes
    25E3No cilia16BLD11Exon 133/33/3Yes
    26D11n/a17Cre17.g7355505′ UTR10/10n/aYes
    17Cre17.g736000Exon 510/10n/aYes
    27F3n/a6Cre06.g266450Intron 311/14n/aNo
    6Cre06.g263650Exon 1314/14n/aYes
    29D5n/a2Cre02.g0863005′ UTR9/9n/aYes
    31D11Multicilia11SOUL3′ UTR3/3n/aYes
    16BAR1Intron 596/96b96/96bYes
    17Chr17-4.678Mn/a1/3n/aNo
    32F1Paralyzed cilia1DZIP1LExon 1812/1212/12Yes
    36A12n/a5Cre05.g2428503′ UTR10/10n/aYes
    6Cre06.g2781075′ UTR10/10n/aYes
    39A9n/a13Cre13.g589450Intron 11/6n/aNo
    17No predicted genen/a6/6n/aYes
    53F8No cilia2Cre02.g145950Exons 5 to 8 + introns 5 to 78/88/8Yes
    2PTK243′ UTR8/88/8Yes
    4SUB7Introns 1 and 2 + exon 23/83/8No
    12No predicted genen/a8/88/8Yes
    • ↵a Number of complete tetrads.

    • ↵b Number of random progeny. n/a not available

Additional Files

  • Figures
  • Tables
  • Supplemental Data

    Supplemental Tables and Data

    Files in this Data Supplement:

    • Supplemental Data - Supplemental Tables 1-6 and Supplemental Data 1-3
PreviousNext
Back to top

Table of Contents

Print
Download PDF
Email Article

Thank you for your interest in spreading the word on Plant Physiology.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
MAPINS, a Highly Efficient Detection Method That Identifies Insertional Mutations and Complex DNA Rearrangements
(Your Name) has sent you a message from Plant Physiology
(Your Name) thought you would like to see the Plant Physiology web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
MAPINS, a Highly Efficient Detection Method That Identifies Insertional Mutations and Complex DNA Rearrangements
Huawen Lin, Paul F. Cliften, Susan K. Dutcher
Plant Physiology Dec 2018, 178 (4) 1436-1447; DOI: 10.1104/pp.18.00474

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Request Permissions
Share
MAPINS, a Highly Efficient Detection Method That Identifies Insertional Mutations and Complex DNA Rearrangements
Huawen Lin, Paul F. Cliften, Susan K. Dutcher
Plant Physiology Dec 2018, 178 (4) 1436-1447; DOI: 10.1104/pp.18.00474
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Extras

  • First author profile: Huawen Lin

Jump to section

  • Article
    • Abstract
    • RESULTS
    • DISCUSSION
    • MATERIALS AND METHODS
    • Acknowledgments
    • Footnotes
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

In this issue

Plant Physiology: 178 (4)
Plant Physiology
Vol. 178, Issue 4
Dec 2018
  • Table of Contents
  • Table of Contents (PDF)
  • Cover (PDF)
  • About the Cover
  • Index by author
View this article with LENS

More in this TOC Section

  • Rapid Affinity Purification of Tagged Plant Mitochondria (Mito-AP) for Metabolome and Proteome Analyses
  • An Online Database for Exploring Over 2,000 Arabidopsis Small RNA Libraries
  • Rapid Single-Step Affinity Purification of HA-Tagged Plant Mitochondria
Show more BREAKTHROUGH TECHNOLOGIES

Similar Articles

Our Content

  • Home
  • Current Issue
  • Plant Physiology Preview
  • Archive
  • Focus Collections
  • Classic Collections
  • The Plant Cell
  • Plant Direct
  • Plantae
  • ASPB

For Authors

  • Instructions
  • Submit a Manuscript
  • Editorial Board and Staff
  • Policies
  • Recognizing our Authors

For Reviewers

  • Instructions
  • Journal Miles
  • Policies

Other Services

  • Permissions
  • Librarian resources
  • Advertise in our journals
  • Alerts
  • RSS Feeds

Copyright © 2021 by The American Society of Plant Biologists

Powered by HighWire